Langmuir 1990,6, 1551-1557
1551
Orientation of a Tripeptide on Platinum Hans Arwin,*i+Ingemar Lundstrom,f Sal0 Arielly,* and Goran Claesons Laboratory of Applied Physics, Department of Physics and Measurement Technology, Linkoping, Institute of Technology, S-581 83 Linkoping, Sweden, Kabi Diagnostika, Talieghrdsgatan 3, S-431 33 Molndal, Sweden, and Kabi AB, Lindhagensgatan 133, S-112 87 Stockholm, Sweden Received December 5, 1989. I n Final Form: April 20, 1990 Capacitance measurements and ellipsometry have been combined to measure adsorption isotherms on platinum electrodes for the tripeptide Bz-Phe-Val- Arg-pNa and some related compounds. The peptides adsorb essentially reversibly but with a small hysterisis. The area each molecule occupies on the surface depends on the surface concentration but not on the molecular structure. The results show that the aromatic rings are essential for the adsorption. A higher surface affinity is obtained if the benzene rings of the peptides have NO2 groups. We discuss mechanisms of adsorption in terms of molecular structure, molecular orientation, and conformation. Introduction When organic molecules adsorb or bind to surfaces, many different physical mechanisms may be responsible for the interaction. For t h e solid/liquid interface, these mechanisms are rather complicated, especially for larger molecules such as proteins. For most proteins, adsorption phenomena are observed, but still many of the details are unknown.' The lack of powerful spectroscopic tools for analyzing thin layers on surfaces in liquids is a limiting factor. Our approach to study surface molecular dynamics is to utilize a combination of two measurement techniques together with chemical modification of the organic molecules. The organic molecules used in this investigation are derivatives of the tripeptide x-Phe-Val-Arg-y, where x can be benzoyl (Bz) or N02-Bz and y can be p-nitroaniline (pNA) or anilide. These small peptides belong to a class of substrates normally used for the assay of enzymatic activity by means of hydrolytic cleavage studied with spectrophotometric techniques. However, we have found that these substrates readily adsorb reversibly to metal surfaces. The adsorption gives rise to large changes in the electrical properties of the metal/solution interface.2 The kinetics and isotherms of the adsorption are very informativeq3v4 The substrate Bz-Phe-Val-Arg-pNA has also been used in the development of an electrode adsorption method for the detection and quantification of serine protease^.^-^ The electrode adsorption method relies on the fact that the tripeptide adsorbs reversibly and that the products formed in the hydrolytic cleavage do not adsorb to any large extent. In our early work with the tripeptides, we found that the aromatic rings and their substituents to a large extent determine the adsorption properties. The fun+ Linkoping Institute of
Technology.
Kabi Diagnostika. I Kabi AB. Now working for Thrombosis Research Institute, Manresa Building, Manresa Road, London SW3 6LR, U.K. t
(1) Horbett, T. A.; Brash, J. L. In Proteins at Interfaces, Physicochemical and Biochemical Studies; Brash, J. L.; Horbett, T. A,, Eds.; ACS Sympium Series 343; American Chemical Society: Washington, DC 1987;
P 1.
(2) Arwin, H.; Lundstrom, I. Reo. Sci. Instrum. 1976, 47, 1394. (3) Arwin, H.; Lundstrom, I. Surf. Sci. 1984, 140, 339. (4) Arwin, H.; Lundstrom, I. Surf.Sci. 1984, 140, 321. (5) Arwin, H.; Lundstrom, I. FEBS Lett. 1980,109, 252. (6)Arwin, H.; Lundstrom, I.; Claeson, G.; Ekenstam, B.; Aurell, L. FEBS Lett. 1976, 64, 95.
(7) Agner, E.; Claeson, G.; Palmqvist, A.; Arwin, H.; Lundstrom, I. J. Biochem. Biophys. Meth. 1983,8, 69.
0743-7463/90/2406-1551$02.50/0
damental questions we addressed were: Why does the substrate Bz-Phe-Val-Arg-pNA adsorb onto several metals? How can the adsorption be improved? Can the adsorption properties be transferred to other molecules? In this communication, we present ellipsometric and capacitance measurements on the parent tripeptide BzPhe-Val-Arg-pNA and modifications thereof. As the peptides are specially prepared for this work, we shortly describe their synthesis. The experimental techniques are only briefly described. The results are interpreted in models developed in our earlier ~ o r k . Finally, ~ ? ~ the adsorption properties are discussed in terms of the difference in molecular structure between the different peptides. Materials and Methods Synthesis of Peptides. The synthesis of the five peptides in Table I is described below. The following abbreviations are used: DMF = dimethylformamide, Bz = benzoyl, pNA = p-nitroaniline, EtOAc = ethyl acetate, HOBT = l-hydroxybenzotriazole, DCC = N,N1-dicyclohexylcarbodiimide,TEA = triethylamine, OSu = N-hydroxysuccinimideester, Cbo = (benzyloxy)carbonyl,and Tris = tris(hydroxymethy1)aminomethane. To characterize the substances obtained, thin-layer chromatograms (TLC) were run on precoated silica gel plates (60 F Merck) in the following solvent systems (ratio by volume): A = 1-butanol-acetic acid-Hz0 (3:1:1),PQ = chloroform-methanolH2O-acetic acid (34:4:2:9), and PI = chloroform-methanol (9: 1). Spots were revealed by their UV absorption (254 nm) and by their reaction to ninhydrin and dicarboxydin. I. Peptide A: Bz-Phe(pNOz)-Val-Arg-pNA.HCI. This compound was prepared as described by Takada et al? [ a ] 2 5 ~ = -42.5' ( C = 0.5431, 50% AcOH). 11. Peptide B: pNO2-Bz-Phe-Val-Arg-pNA.HF. a. pNOz-Bz-Phe-Val-Arg(NO2)-pNA. p-Nitrobenzoyl chloride (100 mg, 0.54 mmol) was added to a solution of H-Phe-Val-Arg(NOz)-pNA.HBr (300 mg, 0.4 mmol) in pyridine (5 mL), prepared as described by Blomback et al.9 The mixture was stirred for 2 h at room temperature, precipitated with ether, and filtered. The precipitate was washed with ether and Hz0. The crude substance was recrystallized from methanol-ether: yield 165 mg (5070); R,(A) = 0.89. b. pNOz-Bz-Phe-Val-Arg-pNA.HF. Anisol (0.2 mL) and liquid HF (10 mL) were added to 147 mg (0.2 mmol) of the peptide described in 1I.a above. The mixture was stirred for 45 min at 0 'C followed by evaporation of the HF in vacuo. The resi(8)Takada, K.; Sakakibara,S.; Kato, H.; Goto, T.; Iwanaga, S. Thrombosis Res. 1980, 20, 533. (9) Blomback, G. E. B.; Blomback, M.; Claeson, K. G.; Svendsen, L. G. English Patent No. 1428039.
0 1990 American Chemical Society
1552 Langmuir, Vol. 6, No. 10, 1990 Table I. Structure and Molecular Weight of the Tripeptides Used in the Measurements. peptide structure MWb A Bz-Phe@NOz)-Val-Arg-pNA.HCI 726.2 B 02N-Bz-Phe-Val- Arg-pNAaHF 709.7 C Bz-Phe-Val- Arg-pNAmHCl 681.2 D 02N-Bz-Phe-Val-Arg-anilideHF 664.7 E Bz-Phe-Val-Arg-anilide-HF 619.7
Arwin et al. Five MWc 689.7 689.7 644.7 644.7 599.7
Note that peptides A and B are very similar, since the amino acid Phe has a benzene ring in its side chain. Molecular weight of the peptide for the salt given in the column to the left. Molecular weight of the HC1 salt of the peptide in solution. Lamp
due was crystallized from methanol-ether: yield 115 mg (81901; R,(A) = 0.57, RAP%) = 0.48; [ a ] %= ~-47.4" (c = 0.5%, 50% acetic acid). 111. P e p t i d e C: Bz-Phe-Val-Arg-pNA.HC1. This compound was prepared as described by Blomback et aL9 [cY]'~D = -43.5' ( C = 0.69'( , 50r' AcOH). IV. Peptide D: pN02-Bz-Phe-Val-Arg-Anilide-HF. a. Cbo-Arg(NO&Anilide. This compound was prepared as described by Blomback e t al.9 for Cbo-Arg(N02)-pNA, using aniline instead of p-nitroaniline: yield 61 r( ; R,(P,) = 0.35; mp 168-169 "C. b. Cbo-Phe-Val-OH. A solution of 3.96 g (10 mmol) of CboPhe-OSu, prepared as described by Andersson et al.1° in 50 mL of dioxan was added to a solution of 1.17 g (10 mmol) of ValOH in 50 mL of water containing 1.68 g (20 mmol) of sodium bicarbonate at 4 "C. The suspension was stirred for 2 h at room temperature. The solution was concentrated in vacuo. Acidification to pH 2 with l o r r HCl gave an oil which crystallized: yield 3.2 g (81 ); R,(A) = 0.8. c. Cbo-Phe-Val-Arg(NO&Anilide. HBr (8 mL, 5.6 M acetic acid) was added to substance IVa (2.14 g, 5 mmol) in glacial acetic acid (10 mL). The mixture was stirred for 30 min a t room temperature, precipitated with ether, and filtered. The precipitate was washed with ether and dried in vacuo. The substance was dissolved in DMF (40 mL) and neutralized a t -10 "C with TEA (1.05 mL, 7.5 mmol). T o this mixture, substance IVb (1.99 g, 5 mmol), HOBT (675 mg, 5 mmol), and DCC (1.03 g, 5 mmol) were added. After 18 h at room temperature, the suspension was filtered and the solvent removed in vacuo. The residue was dissolved in EtOAc and washed with 2? NaHC03, water, 0.4 N HC1, and water. The substance precipitated during the washing, filtered, and washed with a little EtOAc and ether: yield 1.88 g (56'0); R,(A) = 0.87. d. pNO2-Bz-Phe-Val-Arg(N02)-Anilide. This product was prepared as described under 1I.a above, using H-Phe-ValArg(NO&anilide-HBr (see Va): yield 54$ ; Rf(P1) = 0.52. e. pN02-Bz-Phe-Val-Arg-Anilide.HF.This product was prepared as described under 1I.b above: yield 74'; ; R,(A) = 0.75; [@Iz4D = -73.9" (c = 0.35%, 50% acetic acid). V. Peptide E: Bz-Phe-Val-Arg-AnilideHF. a. Bz-PheVal-Arg-(NO&Anilide. HBr (8 mL, 5.6 M acetic acid) was added to peptide 1V.c (3.37 g, 5 mmol) in glacial acetic acid (10 mL). The mixture was stirred for 30 min a t room temperature, precipitated with ether, and filtered. The precipitate was washed with ether and dried in vacuo. To the substance in pyridine (40 mL), benzoic anhydride (1.35 g, 6 mmol) was added. The obtained mixture was stirred for 2 h, concentrated in vacuo, and precipitated with water. The crude product was dissolved in MeOH and precipitated with ether: yield 2.57 g (8Or
